This application addresses broad Challenge Area (14) Stem Cells, and specific Challenge Topics 14-HL- 102: Bio-models and scaffolds for blood cell production and tissue regeneration and 14-DK-102: Discovery of methods to program stem or progenitor cells. One of the major obstacles facing tissue transplantation is the issue of histocompatibility between donor and recipient. Apart from the unique situation where identical twins are donor-recipient pairs, the only approach to this problem currently in routine clinical use is immunosuppressive therapy to limit graft rejection and graftversus- host disease (GVHD). This therapy is accompanied by generalized immune deficiency as well as organ toxicity. In addition, the need to closely match donors and recipients severely limits the availability of organs for transplantation. This proposal seeks to develop the ability to produce cells of the human immune system in culture. We have discovered culture conditions that allow us to produce T lymphocytes from uncommitted mouse stem and progenitor cells. This technology would provide a means of engineering the immune system to tolerate tissue allografts. In addition, the ability to produce T cells with defined specificities could lead to engineering of immune responses directed against tumors. We propose two specific aims. In the first, we will extend existing studies in our mouse model to fully define the culture conditions required to promote efficient T cell differentiation in culture. We will test the hypothesis that the normal selective process that achieves T cell specificity can be manipulated by including cells expressing relevant histocompatibility antigens in the culture, and by adjusting the responsiveness of the developing T cells using exogenous chemical modulators of T cell activation. The second aim will translate this information to a human context, using bone marrow, cord blood, and human embryonic stem cells as sources of stem and progenitor cells. We will establish efficacy by transplanting engineered human T cells into immunodeficient mice and will monitor xenoreactivity and the ability to promote an immune response to immunization. These basic science studies will be important to establish the feasibility of T cell engineering for a variety of clinical applications. PUBLIC HEALTH RELEVANCE: One of the major obstacles facing the field of tissue engineering is the problem of immune responses against transplanted tissues. This project proposes to define culture conditions that allow engineering of the human immune response to provide normal immune function in the absence of tissue rejection. The proposal will test the ability of human bone marrow, cord blood, and embryonic stem cells to develop into T lymphocytes with normal immune function but lacking reactivity against transplanted tissue.